The technology of genetic engineering allows the transfer of genetic traits between species and permits, in particular, the transfer of enzymes from one species to others. These techniques have first reached commercialization in connection with high-value added products such as pharmaceuticals. The techniques of genetic engineering are equally applicable and cost effective when applied to genes and enzymes which can be used to make basic chemical feedstocks.
A metabolic pathway of interest exists in the bacteria Klebsiella pneumoniae, which has the ability to biologically produce 3-hydroxypropionaldehyde from glycerol. Native microorganisms have the ability to produce 1,3-propanediol from glycerol as well. Commercial interests are exploring the production of 1,3-propanediol from glycerol or glucose, in recombinant organisms which have been engineered to express the enzymes necessary for 1,3-propanediol production from other organisms.
3-hydroxypropionic acid CAS registry Number [503-66-2] (abbreviated as 3-HP) is a three carbon non-chiral organic molecule. The IUPAC nomenclature name for this molecule is propionic acid 3-hydroxy. It is also known as 3-hydroxypropionate, β-hydroxpropionic acid, β-hydroxypropionate, 3-hydroxypropionic acid, 3-hydroxypropanoate, hydracrylic acid, ethylene lactic acid, β-lactic acid and 2-deoxyglceric acid. Applications of 3-HP include the manufacture of absorbable prosthetic devices and surgical sutures, incorporation into beta-lactams, production of acrylic acid, formation of trifluromethylated alcohols or diols, polyhydroxyalkonates, and co-polymers with lactic acid. 3-HP for commercial use is now commonly produced by organic chemical syntheses. The 3-HP produced and sold by these methods is relatively expensive, and it would be cost prohibitive to use it for the production of monomers for polymer production. As discussed below, some organisms are known to produce 3-HP. However, there is not yet available a catalog of genes from these organisms and thus the ability to synthesize 3-HP using the enzymes natively responsible for the synthesis of that molecule in the native hosts which produce it does not now exist.
In addition to its commercial utility, 3-HP it is found in a number of biological processes, notably including many naturally occurring bio-polymers. Poly(3-hydroxybutyrate) (PHB) is the most abundant member of the microbial polyesters which contain hydroxy monomers termed polyhydroxyalkonates (PHAs). PHB has utility as a biodegradable thermoplastic material and the material was first produced industrially in 1982.
The majority of published research on PHA's that contain 3-HP has concentrated on two bacterial sources: Ralstonia eutropha (“Alcaligenes eutrophus”) and Pseudomonas oleovorans. Both Ralstonia eutropha and Pseudomonas oleovorans are able to grow on a nitrogen free media containing 3-hydroxy -propionic acid, 1,5-pentanediol or 1,7-heptanediol. When 3-HP is the major hydroxy-acid added to the growth media, poly(3-hydroxybutyrate-co-3-hydroxypropionic acid) is formed containing 7 mol % 3-hydroxypropionic acid. These cells also store 3 mol %, 3-hydroxypropionic acid poly(3-butyrate-co-3-hydroxypropionic acid).
Recombinant systems have been used to create PHAs. An E. coil strain engineered to express PHA synthase from either Ralstonia eutropha or Zoolgoea ramigera produced poly(3-hydroxypropionic acid) when feed 1,3-propanediol. Skraly, F. A. “Polyhydroxyalkonates Produced by Recombinant E. coli.” Poster at Engineering Foundation Conference: Metabolic Engineering II, 1998. An E. coli strain that expressed PHA synthase (MBX820), when provided with the genes encoding glycerol dehydratase and 1,3-propanediol dehydratase from K. pneumonia, and 4-hydroxybutyral-CoA transferase from Clostridium kluyveri, synthesized PHB from glucose.
Glycerol dehydratase, found in the bacterial pathway for the conversion of glycerol to 1,3-propanediol, catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde and water. This enzyme has been found in a number of bacteria including strains of Citrobacter, Klebsiella, Lactobacillus, Entrobacter and Clostridium. In the 1,3-propanediol pathway a second enzyme 1,3-propanediol oxido-reductase (EC 1.1.202) reduces 3-hydroxypropanaldehyde to 1,3-propanediol in a NADH dependant reaction. The pathway for the conversion of glycerol to 1,3-propanediol has been expressed in E. coli. Tong et al., Applied and Environmental Microbiology 57 (12)3541-3546. The genes responsible for the production of 1,3-propanediol were cloned from the dha regulon of Klebsiella pneumoniae. Glycerol is transported into the cell by the glycerol facilitator, and then converted into 3-hydroxy-propionaldehyde by a coenzyme B12-dependent dehydratase. E. coli lacks a native dha regulon, consequently E. coli cannot grow aerobically on glycerol without an exogenous electron acceptor such as nitrate or fumarate.
Aldehyde dehydrogenases are enzymes that catalyze the oxidation of aldehydes to carboxylic acids. The genes encoding non-specific aldehyde dehydrogenases have been identified in a wide variety of organisms e.g.; ALDH2 from Homo sapiens, ALD4 from Saccharomyces cerevisiae, and from E. coli both aldA and aldB, to name a few. These enzymes are classified by co-factor usage, most require either AND+, or NADP+ and some will use either co-factor. The genes singled out for mention here are able to act on a number of different aldehydes and it likely that they may be able to oxidize 3-hydroxy-propionaldehyde to 3-hydroxypropionic acid.